Upregulation of vitamin D binding protein (Gc-globulin) binding sites during neutrophil activation from a latent reservoir in azurophil granules.

Vitamin D binding protein (DBP) is a multifunctional plasma transport protein that is also found on the surface of many cell types. Cell surface DBP significantly enhances chemotactic activity of complement (C) peptides C5a and C5a des Arg. However, both DBP binding and C5a chemotaxis enhancement can vary among neutrophil donors. To test if activation during cell purification is responsible for this variability, neutrophils were isolated using both standard and lipopolysaccharide (LPS)-free protocols. Cells isolated by the LPS-free method had no DBP-enhanced chemotaxis to C5a or DBP binding to plasma membranes. Moreover, neutrophils treated with LPS bound more avidity to immobilized DBP than sham-treated cells. Subcellular fractionation of neutrophils (standard protocol) revealed a heavy plasma membrane (HM) band that contained components of light plasma membranes and all three granules. The HM band possessed most of the DBP binding activity (58%), and activation of cells with ionomycin greatly increased DBP binding to HM. Azurophil granules contained 33% of the total DBP binding sites and there was a highly significant positive correlation (r=0.988) between release of the granule marker myeloperoxidase and DBP binding. These results indicate that fusion of granules with the plasma membrane forms HM that contains DBP binding sites.

CD44 and annexin A2 mediate the C5a chemotactic cofactor function of the vitamin D binding protein.

The vitamin D binding protein (DBP) is a plasma protein that significantly enhances the chemotactic activity of C5a and C5a(desArg) (cochemotactic activity). The objective of this study was to investigate how DBP mediates this process using neutrophils and U937 cells transfected with the C5a receptor (U937-C5aR cells) and comparing chemotaxis to C-activated serum (DBP dependent) vs purified C5a (DBP independent). Binding to the cell surface is essential for this protein to function as a chemotactic cofactor, and DBP binds to a chondroitin sulfate proteoglycan (CSPG) on neutrophil plasma membrane preparations. To determine whether a CSPG also functions to mediate cochemotactic activity, U937-C5aR cells were grown in chlorate to inhibit CSPG sulfation or treated with chondroitinase AC. Either treatment significantly inhibited chemotaxis only to C-activated serum. CD44 is a major cell surface CSPG on leukocytes, and functions to facilitate chemotaxis. Treatment of cells with anti-CD44 blocks chemotaxis of neutrophils and U937-C5aR cells to C-activated serum but not purified C5a. DBP binds to CD44 on the cell surface as evidenced by coimmunoprecipitation, confocal microscopy, and cell binding studies. Annexin A2 associates with CD44 in lipid rafts; therefore, its potential role in mediating cochemotactic activity was investigated. Results demonstrate that anti-A2 inhibits neutrophil and U937-C5aR chemotaxis specifically to C-activated serum, blocks DBP binding to cells, and colocalizes with anti-DBP on the cell surface. These results provide clear evidence that CD44 and annexin A2 mediate the C5a chemotactic cofactor function of DBP.